Blood DNA Extraction Kit

Transcription

1 H A N D B O O K Blood DNA Extraction Kit NP-BD Preps

2 Contents Page No COMPONENTS Kit Contents Reagents, Consumables and Equipment not provided with the kit SAFETY INSTRUCTIONS INTRODUCTION Principle and Procedure Preparation and Storage of reagents PROTOCOL FOR PURIFICATION OF GENOMIC DNA Genomic DNA Extraction from Whole Blood Genomic DNA Extraction from Large Blood Genomic DNA Extraction from Nucleated Blood Genomic DNA Extraction from Bone Marrow. Genomic DNA Extraction from Buffy Coat TROUBLESHOOTING GUIDE PRODUCT WARRANTY COMPONENTS Kit contents Blood DNA Extraction Kit Cat # NP-BD-050 NP-BD-050 No. of Preps (50 preps.) (250 preps.) Proteinase K solution 1.2ml 5 X 1.2ml Lysis Buffer BLB 24ml 120ml Wash Buffer (conc) BWB1 10ml 40ml Wash Buffer (conc) BWB2 10ml 40ml Elution Buffer BEB 30 ml 150ml Spin Columns Collection Tubes Handbook 1 1 *Please see Preparation and Storage of reagents.

3 Reagents, consumables and equipments not providedwith the kit Ethanol Microcentrifuge Water Bath or Heat Bath,etc SAFETY INSTRUCTIONS When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDS). INTRODUCTION Principle and Procedure Blood DNA Extraction Kit is designed for rapid and efficient purification of high quality genomic DNA from whole blood and related body fluids. The kit utilizes silica-based membrane technology in the form of a convenient spin column, eliminating the need for expensive resins, toxic phenol-chloroform extractions, or time consuming alcohol precipitation. The standard procedure takes less than 20 minutes following cell lysis and yields purified DNA greater than 30 kb in size. Isolated DNA can be used directly in PCR, qpcr, Southern blotting and enzymatic reactions. Preparation and Storage of reagents Blood DNA Extraction Kit should be stored dry at room temperature (18 26 C). Kits can be stored for up to 12 months without showing any reduction in performance and quality. Proteinase K solutions are stable at room temperature as long as not opened. After being opened they should be stored at -20 C. Other components of the kit should be stored at room temperature (18 26 C). Add 30ml ethanol (96-100%) to Wash Buffer BWB1 (NP-BD-050) and 120ml ethanol (96-100%) to Wash Buffer BWB1(NP-BD-250 ) (concentrated) prior to first use. Add 30ml ethanol (96-100%) to Wash Buffer BWB2(NP-BD-050 ) and 120ml ethanol (96-100%) to Wash Buffer BWB2 (NP-BD-250 ) (concentrated) prior to first use. Check the Lysis Buffer BLB for salt precipitation before each use. Re-dissolve any precipitate by warming the solution at 37 C, then cool back down to 25 C before use.

4 Protocol for Purification of Genomic DNA Things to do before starting Check if Proteinase K & Wash Buffers were prepared as per instructions (A) Blood DNA Extraction Protocol 1. Add 20 µl of Proteinase K Solution to 200 µl of whole blood, mix by vortexing. Add 400 µl of Lysis Buffer BLB, mix thoroughly by vortexing or pipetting to obtain a uniform suspension. Note. If using less than 200 µl of blood, adjust sample volume to 200 µl with 1X PBS or TE buffer (not provided). 2. Incubate the sample at 56 C for 10 minutes while vortexing occasionally or use a shaking water bath, rocking platform or thermomixer until the cells are completely lyse. 3. Add 200 µl of ethanol (96-100%) and mix by pipetting. 4. Transfer the prepared mixture to the spin column. Centrifuge for 1 min at 6,000 g (~8,000 rpm). Discard the collection tube containing the flow-through solution. Place the column into a new 2 ml collection tube (included). Important: do not exceed specified relative centrifugal force. 5. Add 500 µl of Wash Buffer BWB1. Centrifuge for 1 min at 8,000 g (~10,000 rpm). Discard the flow-through and place the column back into the collection tube. 6. Add 500 µl of Wash Buffer BWB2 to the column. Centrifuge for 3 min at maximum speed ( 20,000 g, 14,000 rpm). Recommended: Empty the collection tube. Place the purification column back into the tube and re-spin the column for 1 min. at maximum speed ( 20,000 g, 14,000 rpm). Discard the collection tube containing the flow-through solution and transfer the column to a sterile 1.5 ml microcentrifuge tube (not included).

5 7. Add 200 µl of Elution Buffer BEB to the center of the column membrane to elute genomic DNA. Incubate for 2 min at room temperature and centrifuge for 1 min at 8,000 g (~10,000 rpm). Note For maximum DNA yield, repeat the elution step with an additional 200 µl of Elution Buffer. If more concentrated DNA is required or if DNA has been isolated from a small amount of starting material (e.g., 50 µl) the volume of the Elution Buffer added to the column can be reduced to µl. Please be aware that lower volumes of Elution Buffer will result in lower final yield of eluted DNA. 8. Discard the purification column. Use the purified DNA immediately in downstream applications or store at -20 C.. (B) Large volume Blood Genomic DNA Purification Protocol DNA Purification from Large Volumes of Whole Blood For purification of DNA from samples exceeding the standard 200 µl volume, it is necessary to burst red blood cells prior to performing the cell lysis step. Up to 500 µl of mammalian blood can be processed using following protocol. 1. Add 1 ml of ice cold nuclease free water to 500 µl of whole blood, mix thoroughly by vortexing or pipetting. 2. Incubate the sample for 5 min at room temperature. 3. Centrifuge for 5 min at 800 g (~3,000 rpm). Discard the supernatant. 4. Resuspend the pellet in 200 µl of 1 x PBS. 5. Proceed to step 1 of the Blood DNA extraction standard protocol. (C) Genomic DNA Purification Protocol from Nucleated Blood Nucleated avian or fish blood contains very large amounts of genomic DNA and therefore the volume of the staring material has to be scaled down. The DNA purification procedure follows the same protocol as mammalian blood, except that 2-10 µl of blood are used per purification. 1. Take 2-10 µl of nucleated blood. 2. Adjust the volume to 200 µl with 1 PBS.

6 3. Proceed to step 1 of the Blood DNA extraction standard protocol (D) Genomic DNA Purification Protocol from Bone Marrow 1. Harvest µl of fresh or frozen bone marrow. 2. Adjust the volume to 200 µl with 1 PBS. 3. Proceed to step 1 of the Blood DNA extraction standard protocol (E) Genomic DNA Purification Protocol from Buffy Coat Buffy coat is a leukocyte-enriched fraction of whole blood and contains approximately 5-10 times more DNA than an equivalent volume of whole blood. Prepare the buffy coat by centrifuging whole blood at 2,500 g for 10 min at room temperature. After centrifugation, 3 different fractions are distinguishable: the upper clear layer containing plasma; the intermediate buffy coat layer containing concentrated leukocytes, and the bottom layer containing concentrated erythrocytes 1. Centrifuge 1.5 ml of whole blood at 2,500 g (~5,000 rpm) for 10 minutes at room temperature. Three layers should be visible. 2. Remove upper clear layer by aspiration. 3. Collect approximately 200 µl of intermediate layer using an automatic pipette. Note. If necessary, adjust the volume to 200 µl with 1 PBS. 4. Proceed to step 1 of the Blood DNA extraction standard protocol

7 TROUBLESHOOTING GUIDE Low yield of purified DNA Excess sample used during lysate preparation. Reduce the amount of starting material. Do not use more blood than indicated in lysis protocols. Starting material was not completely digested. Extend the Proteinase K digestion at 56 C until complete lysis occurs and no particles remain visible in solution. Ethanol was not added to the lysate. Ensure that ethanol was added to the lysate before applying the sample to the Purification Column. After the addition of ethanol to the lysate, mix the sample by vortexing or pipetting. DNA is not performing well in subsequent experiments Incorrect volume of Lysis buffer added to the samples Ensure the correct volume of Lysis Buffer has been added to the sample. Also, make sure all centrifugation steps are completed for the indicated times and speeds (rcfs). Failure to do so may result in incomplete washing, which may cause salts to be eluted with the DNA affecting quantitation and subsequent experiments including enzymatic processes like PCR. RNase A contamination RNA contamination The buffers and spin columns provided in this kit are designed to efficiently remove RNA during the DNA purification procedure. However, additional RNA removal (e.g., digestion with RNase A) may be necessary for subsequent applications sensitive to trace amounts of RNA.

8 Genetix Biotech Asia Pvt. Ltd. 71/1, First Floor, Shivaji Marg, Najafgarh Road, New Delhi Phone : Fax : info@genetixbiotech.com